Liposomes as immunological adjuvants in vaccines: studies with entrapped and surface-linked antigen

muscle (Ash & Tucker, 1967) and stimulated a chloridedependent hyperpolarization of individual muscle cells (Del Castillo et al., 1964; Martin, 1980). The properties and reaction mechanism of GABA-T from a nematode parasite of rats (Nippostrongylus brasiliensis) have been described in detail (Watts & Atkins, 1984) and, by analogy with the findings in mammalian brain tissue, it was of interest to discover whether certain specific, mechanism-based, irreversible inhibitors of nematode GABA-T, such as hydrazinopropionic acid (HPA) (Watts & Atkins, 1986), would result in increased GABA levels in whole worms exposed to such compounds in vitro. The possible therapeutic relevance to the development of new anthelmintics is obvious since paralysed parasites would be expected to be purged from the gastrointestinal tract of the host or, in the case of tissuedwelling nematodes, to be rendered less efficient in performing any physiological processes dependent upon GABA for normal muscular control. A recent patent application (Krantz & Castelhano, 1984) describes certain y-alkenyl-yaminobutyric acids, inhibitors of pig brain GABA-T, which paralyse the free-living nematode Caenorhabditis elegans and kill N . hrusiliensis, in vitro. In the present report several irreversible, mechanism-based inhibitors of N . brasiliensis GABA-T are described and their effects examined on GABA levels in whole worms. The preparation of GABA-T from 7-day-old adult N. brasiliensis and the determination of the dissociation constants of the enzyme inhibitor complexes ( K , ) and the irreversible inactivation rate constants (klrc) were performed as described previously (Watts & Atkins, 1986) for HPA. The K, and k,,, values for a 3-pyrazolidinone GABA-T inhibitor, BW356U, for HPA and for gabaculine were, respectively: 1 6 . 2 ~ ~ . 0.0087s ' ; 17.511~. 0.45s I; 3 9 5 n ~ , O.OlIs-'. A radioligand-binding assay was utilized for the determination of GABA levels in N . brasiliensis. This involved measuring the displacement of specific binding of the GABA agonist [3H]muscimol from rat brain GABA receptors by any GABA present in neutralized perchlorate extracts of nematodes. Rat brain receptors were prepared essentially according to the procedure of Zukin et al. (1974) and the muscimol-binding experiments were performed in a manner similar to that of Leach & Wilson (1978) with calf brain receptors. The proportion of total muscimol binding due to the specific component was derived from incubations which included 0.25 mM-GABA to eliminate, by competition, all specific binding due to the 5 nM-muscimol present in incubations. No interference with the binding assay was caused by the neutralized perchlorate extracts of N. brasiliensis; the predicted inhibition of muscimol binding due to GABA was evident in samples of extract to which known amounts of GABA were added. The absolute concentration of GABA in a given sample was calculated by fitting the observed inhibition to the equation of the line for the GABA calibration standards. Triplicate determinations were performed on each batch of parasites. The overall mean for unincubated, untreated worms was 0.476 0.092 pmol of GABA per worm; overnight incubation of worms, in vitro, did not effect this value significantly. The effects of GABA-T inhibitors on GABA levels in N. brasiliensis were tested, in vitro, by incubating approximately l00mg wet weight of parasites for 16h in the following solution, with and without 1 .O mM-inhibitor: 4.5 mMK,HPO,, 5.8 mM-KH,PO,, 27 mM-D-glucose, 2 g of tryptic digest of casein (Difco), 1 g of yeast extract (Difco) adjusted to pH 7.2 with KOH and autoclaved to sterilize before addition of 78 p~-5-fluorocytosine, 100 000 i.u. of sodium benzyl penicillin and 100 mg of streptomycin sulphate/ l00ml. Incubations were terminated by filtration and washing in phosphate buffer. Parasites were removed from the filter and homogenized in 0.9 M-perchloric acid before a low-speed centrifugation to separate cell debris. This debris was re-extracted with 0.2 M-perchlorate and the combined supernatants neutralized with KOH (Methyl Red indicator). Samples of the neutralized extract were used for the GABA assay. The known GABA-T inhibitors resulted in increases in the GABA concentration of whole nematodes over control parasites incubated without drug of 70% for BW365U. 186% for HAP and 415% for gabaculine. The non-specific inhibitor of pyridoxal phosphate-linked enzymes, aminooxyacetate, resulted in only a 12% increase in GABA at 1 .O mM. This report represents, therefore, the first demonstration that GABA levels in nematodes can be manipulated by appropriate treatment of whole parasites with inhibitors of isolated enzymes. Whether it will be possible to translate these observations into a chemotherapeutic effect in vivo depends upon the pharmacokinetics of parasite exposure to the compounds in question and on the unknown longer-term mechanisms controlling GABA levels around synapses in nematodes.